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Carbohydrate Polymers

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Multifunctional prosthetic polyester-based hybrid mesh for repairing ofabdominal wall hernias and defects

Mohamed M. Shokrya, Islam A. Khalilb,c,1, Abdelhaleem El-Kasapyd, Ahmed Osmane,Ayman Mostafaa, Mohamed Salahf, Ibrahim M. El-Sherbinyb,⁎

a Department of Surgery, Anesthesiology and Radiology, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, EgyptbNanomaterials & Nanomedicine Lab, Center of Material Science (CMS), Zewail City of Science and Technology, 6th of October, Giza, 12588, Egyptc Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy and Drug Manufacturing, Misr University of Science and Technology (MUST), 6thofOctober, Giza, 12566, EgyptdDepartment of Surgery, Faculty of Veterinary Medicine, Benha University, Moshtohor, Egypte Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, EgyptfDepartment of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Cairo University, Giza, 12211, Egypt

A R T I C L E I N F O

Keywords:PhenytoinCiprofloxacinChitosanPolyester fabricNanocarrierProsthetic meshHernia

A B S T R A C T

This study involves the design, development and evaluation of a new multifunctional prosthetic mesh fortreatment of abdominal wall defects without complications. The developed prosthetic mesh is a hybrid platformof both synthetic and natural materials with its backbone consisting of a synthetic commercial polyester fabric(CPF) to provide the required mechanical integrity. The CPF mesh was coated by a natural biodegradable,biocompatible and antimicrobial layer of chitosan (CS) incorporating phenytoin (PH)-loaded pluronic nano-micelles for healing promotion, and ciprofloxacin (CPX)-alginate polyelectrolyte complex-based microparticlesas antibacterial agent. The prosthetic mesh was optimized and evaluated in-vitro and in-vivo. The optimum PH-loaded micelles had particle size of 95.42 nm, polydispersity index of 0.41, zeta potential of -18 and entrapmentefficiency of 89.4%, while the optimum CPX microcomplexes had particle size of 1292.0 nm, polydispersityindex of 0.8, zeta potential of -20.1, complexation efficiency of 81.1%, and minimum inhibitory concentration of0.25 μg/ml and 0.125 μg/ml against Staphylococcus aureus and Pseudomonas aeruginosa, respectively. In-vivostudy on abdominal wall defect dog model was conducted, followed by implantation of the proposed prostheticmeshes. The developed mesh depicted an efficient healing with excellent biocompatibility, and could be an idealand feasible alternative prosthesis with many advantages such as low cost, inertness, mechanical stability,pliability, low infection rate, limited modification by body tissues, sterilizability, non-carcinogenicity, limitedinflammatory reaction, hypoallergenic as well as minimal complications.

1. Introduction

Hernia is a term used to describe a case, where part of internal organis displaced and protrudes through a hole of the containing cavity.There are many types of hernia, depending on place of the defect, suchas inguinal, incisional and umbilical hernias. Hernia repair is one of themost common procedures in surgical practice (Salgaonkar & Lomanto,2017), and the conventional method is suitable only for small herniaring (Brown & Finch, 2010). Currently, hernioplasty, a type of herniarepair surgery where a mesh patch is sewn over the weakened region ofa tissue, represents the standard technique for repairing inguinal herniaworldwide. However, extensive abdominal wall defects with

voluminous hernias still represent a great challenge due to the large sizering with marginal distortion. Furthermore, large defect with tension onthe closure of the wound can affect surgical outcome (Park & Lakes,2007). Therefore, surgeons and researchers still working on developingnew techniques to improve the surgical outcomes.

Prosthetic meshes are usually implanted either intraperitoneally orextraperitoneally (Shoukry, El-Keiey, Hamouda, & Gadallah, 1997),where they can be applied by physical pressure between abdominalwall layers (Stoppa et al., 1984), non-absorbable sutures (Gourgiotiset al., 2006), absorbable material sutures (Gianlupi & Trindade, 2004),clips (Read, 2011) or fibrin glue (Agresta & Bedin, 2008). Moreover,prosthetic meshes should be noncarcinogenic, chemically and

https://doi.org/10.1016/j.carbpol.2019.115027Received 28 May 2019; Received in revised form 19 June 2019; Accepted 24 June 2019

⁎ Corresponding author.E-mail address: ielsherbiny@zewailcity.edu.eg (I.M. El-Sherbiny).

1 Conducted the experiments related to the nano-part.

Carbohydrate Polymers 223 (2019) 115027

Available online 05 July 20190144-8617/ © 2019 Elsevier Ltd. All rights reserved.

T

biologically inert, mechanically stable, sterilizable, biocompatible, hy-poallergenic as well as amenable (Hamer-Hodges & Scott, 1985). Gen-erally, it is not easy to have a mesh that meets all of these features.Several materials have been used to date to fabricate prosthetic meshes.These include, for instance, knitted polypropylene (marlex mesh)(Usher & Gannon, 1959), commercial polyester fabric (Shoukry et al.,1997), nylon, Dacron, stainless steel (Sheen, 2005), cotton mesh,mosquito net mesh (Stephenson & Kingsnorth, 2011), vicryl mesh (Liuet al., 2016), expanded polytetrafluroethylene (Kennedy & Matyas,1994), mersilene mesh, carbon fibers (Mohsina et al., 2014), oxidizedcellulose polyethylene glycol and hylan G-F20 (Altınlı et al., 2011).Many complications could arise from implantation of prosthetic meshessuch as tissue ingrowth, adhesion to adjacent organs, migration orshrinkage, infection and inflammation. These complications could af-fect the fate of implanted mesh. Therefore, many efforts have beenmade to create synthetic meshes with more biologically acceptablematerials such as chitosan that could confer some additional function-ality like antimicrobial activity (Benhabiles et al., 2012; Pereira et al.,2013). This could limit the recurrence in both conventional surgery andlaparoscopic hernia repair approaches (Sebben, Rocha, Von Bahten, &Biondo-simões, 2006). Also, new technologies have been used to de-velop prosthetic meshes from nanostructured materials with enhancedbiological properties (Rajendran, Suriyaprabha, Sutha, Kavitha, &Prabhu, 2016).

The main objective of this study is to design, develop and evaluate anew multifunctional prosthetic mesh for treatment of abdominal walldefects without complications. The developed prosthetic mesh is a hy-brid platform of both synthetic and natural materials (Fig. 1) with itsbackbone consisting of a synthetic polyester fabric to provide the re-quired mechanical integrity. This polyester mesh was coated by a nat-ural biodegradable, biocompatible and antimicrobial layer of chitosan(CS) incorporating nano and microparticles loaded with two differentdrugs. The first drug is phenytoin (PH), which has been loaded intopluronic (PL) nanomicelles. PH has been selected to enhance the rate oftissue granulation and collagen deposition, as previously reported byour group (Ali, Khalil, & El-Sherbiny, 2016). The second drug is ci-profloxacin hydrochloride (CPX), which is antibacterial agent that be-longs to fluoroquinolones group. CPX was electrostatically crosslinkedwith alginate (ALG) chains to form (CPX-ALG) polyelectrolyte com-plexes-based microparticles.

2. Materials and methods

2.1. Materials

Chitosan, CS (low molecular weight), pluronic F−127 (PL), dis-odium hydrogen phosphate, dipotassium hydrogen phosphate and so-dium tripolyphosphate (TPP) were purchased from Sigma-Aldrich inChina and Germany. Alginate (ALG) was purchased from FisherChemicals (Leicestershire, UK). Ciprofloxacin was obtained from

EIPICO pharmaceutical company (Cairo, Egypt). Phenytoin base (PH)was obtained as a gift from El-Nasr Pharmaceutical Co., Egypt. All otherreagents and solvents were of analytical grade and were used withoutfurther purification.

2.2. Development and characterization of PH-loaded nanomicelles

2.2.1. Preparation of PH-loaded nanomicellesPH was incorporated into pluronic (PL) nanomicelles through na-

noprecipitation technique (Ali et al., 2016). Briefly, 10mg PH weredissolved in 10ml of acetone containing PL according to the ratiosshown in Table 1 (P1, P2 and P3). Then, 10ml of distilled water wasadded slowly with stirring, where PH-loaded nanomicelles were formedspontaneously. The resulting PL nanomicelles suspension was left atroom temperature for 4 h under stirring until complete evaporation ofthe residual acetone.

2.2.2. Evaluation of PH-loaded PL nanomicellesParticle size, polydispersity index and zeta potential of the devel-

oped plain and PH-loaded PL nanomicelles were evaluated using dy-namic light scattering (DLS) technique (Zetasizer Nano-ZS, MalvernInstruments, Malvern, UK). Furthermore, the micelles shape was in-vestigated using scanning electron microscopy, SEM (Nona Nano SEM,FEI, USA). Thermal characteristics were evaluated using differentialscanning calorimetry (DSC) analysis (Q20, TA instrument, USA) at arate of 10 °C/min under nitrogen atmosphere (25ml/min) (Khalil, Ali,& El-Sherbiny, 2019). The entrapment efficiency (EE%) and drugloading (LD%) were determined indirectly using UV–vis spectro-photometer (Evolution UV 600, Thermo Scientific, USA) at 258 nm(Alvarado et al., 2015). EE% and LD% were calculated using Eqs. (1)and (2):

Fig. 1. A schematic illustration of the study design.

Table 1Ratios of ingredients in PH-loaded nanomicelles, CPX-ALG microcomplexes andCS films formulations.

F# PH PL CPX ALG CS TPP CPF

PH nanomicelles P1 1 2P2 1 4P3 1 8

CPX complexesmicroparticles

C1 1 1C2 1 2C3 1 4C4 1 8

CS film CPF0 5×12 cmCPF1 1 10 5×12 cmCPF2 1 2 1 10 5×12 cmCPF3 1 8 1 10 5×12 cmCPF4 1 2 1 8 1 10 5×12 cm

PH: phenytoin, PL: pluronic, CPX: ciprofloxacin, ALG: sodium alginate, CS:chitosan, TPP: tripolyphosphate, CPF: commercial polyester fabric.

M.M. Shokry, et al. Carbohydrate Polymers 223 (2019) 115027

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= − ×EE Total PH Free PH Total PH% (( )/ ) 100 (1)

= + ×LD Loaded PH Total Polymer Loaded PH% ( /( )) 100 (2)

2.2.3. In-vitro release studyThe cumulative release of PH from PL nanomicelles was studied

using diffusion method (Kamel, Khalil, Rateb, Elgendy, & Elhawary,2017). Briefly, one ml of PH-loaded nanomicelles was placed in a donorcompartment covered with dialysis bag (Mw cut-off 12 kDa, Severa)and immersed in 10ml 10% hydroalcoholic PBS using ethanol as thereceiver compartment at 37 °C and 50 rpm. At different time points (1,2, 3, 4, 5, 6, 12, 24 and 48 h), 0.5 ml aliquot was withdrawn and re-placed with fresh media (Ali et al., 2016). PH concentration was mea-sured at 258 nm using UV–vis spectrophotometer, and the cumulativeconcentration was calculated using Eq. (3).

∑= +=

−

Cn Cn means A V Cs means/s

n

1

1

(3)

where Cn is the expected nth sample concentration, Cn means is themeasured concentration, A is the volume of withdrawn aliquot, V is thevolume of the dissolution medium, n-1 is the total volume of all thepreviously withdrawn samples before the currently measured sample,and Cs is the total concentration of all previously measured samplesbefore the currently measured one. Different kinetic models were ex-amined to find the best fitting release profile using the excel add-insoftware package, DDSolver (Zhang et al., 2010).

2.3. Development and characterization of CPX-ALG microcomplexes

2.3.1. Preparation of CPX-ALG microcomplexesCPX was dissolved in acidic medium (pH 3) while ALG was dis-

solved in basic medium (pH 9) followed by mixing the two solutionsunder stirring at different ratios (Table 1). The resulting CPX-ALG mi-crocomplexes were centrifuged (Sigma 3-18KS), washed twice withdistilled water and lyophilized.

2.3.2. Evaluation of CPX-ALG microcomplexesParticle size, polydispersity index and zeta potential of the prepared

CPX-ALG microparticles were investigated using dynamic light scat-tering (DLS) technique (Zetasizer Nano-ZS, Malvern Instruments,Malvern, UK). Thermal behavior was tested using DSC analysis.Complexation efficiency was measured indirectly at 277 nm similar toEE%. The release profile of CPX was evaluated using diffusion cell aspreviously described.

2.3.3. In-vitro microbiology studyAntibacterial activity of CPX-ALG microcomplexes was evaluated

through detecting the minimum concentration that inhibits bacterialgrowth (MICs) of both Staphylococcus aureus (ATCC 29,213) andPseudomonas aeruginosa (ATCC 25,175) using a microdilution method(Zhao, Li, Guo, & Ma, 2015). Briefly, bacterial inocula was transferredinto media tube followed by incubation for 24 h at 35 °C. Then, 50 μl ofdifferent CPX concentrations (14–0.014 μg/ml) were placed in 96-wellplate. Afterwards, 150 μl of microorganism suspension (1× 106 CFU/ml; CFU= colony forming units) was added to the drug suspension. Theturbidity was measured at 562 nm after 12 h (log phase). Also, positiveand negative controls were tested by incubating bacteria without drugand broth without bacteria, respectively. All samples were tested intriplicates.

2.4. Preparation of the drugs-loaded CS coating

CS coating layer was obtained using the casting solvent-evaporationtechnique. Briefly, 1% w/v CS solution was prepared in 1% acetic acid,followed by dispersion of the predetermined amounts of PH-loaded PL

nanomicelles or the CPX-ALG microparticles as demonstrated inTable 1. Afterwards, TPP solution was prepared and mixed with the CSsolution at weight ratio of 1:10. Commercial polyester fabric (CPF) wasused as the backbone for the developed prosthetic hernia mesh. CPFwas immersed in fresh drugs-loaded CS/TPP solution. The resulting CS-coated CPF mesh was left to dry overnight. Visual examination of theobtained coating film was performed to check its integrity. Differentcoating films were thermally evaluated using DSC. Moreover, CPF2 wastested, as example, for drug release profile at 258 nm, while CPF3 wastested at 277 nm.

2.5. In-vivo study

2.5.1. Induction of abdominal wall defectThe in-vivo study was approved by the Cairo University Institutional

Animal Care and use ethical Committee Cu-IACUC) (CU/II//39/2017).Twelve mongrel dogs (7 males and 5 females) were used with ages of 1to 3 years, and weighing 15 to 25 kg. The animals were housed in in-dividual box with a supply of water and food ad libitum. Access to foodwas restricted for 12 h before the operation. Drug regimen with ampi-cillin (25mg/kg, i.m.) twice daily started before operation and con-tinued for 7 days postoperatively. Directly before the surgery, xylazine(2.0 mg/kg, i.m., Rompun; Bayer) and ketamine (5.0 mg/kg, i.v.,Ketavet; Parke Davis) were given. The mesogastrium region was pre-pared for aseptic surgery. General anesthesia was induced by oro-tracheal intubation and maintained on isoflurane inhalant anesthesia(Fig. 2a). Dogs were subjected to midline laparotomy incision throughthe linea alba and the rectus abdominis muscle was exposed on bothsides to suitable distance. A serial circumferential row of interruptedstitches using absorbable suture material, enclosing the measured de-fect to minimize hemorrhage, were placed before the induction. Thedefect was then produced bilaterally 2 cm from the linea alba, includingthe muscle layer only in 4 animals and encompassing the peritoneum in12 animals (Fig. 2b). Dogs were allocated to four groups. In each group,one dog was subjected to intraperitoneal implantation (Fig. 2c) and twodogs were subjected to retroperitoneal implantation (Fig. 2d).

2.5.2. Study groupsThe operated dogs were randomly and equally allocated into four

groups of three dogs each in which two dogs with peritoneal defect andone dog without peritoneal defect. Group A: This group was subjectedto standard CPF mesh hernioplasty; Group B: This group was subjectedto CPF1 mesh hernioplasty (non-medicated CS coated mesh); Group C:This group was subjected to CPF3 mesh hernioplasty (antibacterial drugCPX-incorporated CS coated mesh), and Group D: This group wassubjected to CPF4 mesh hernioplasty (both drugs CPX and PH-in-corporated CS coated mesh).

2.5.3. Intraperitoneal mesh hernioplastyThe meshes were folded and trimmed to match the size of the ab-

dominal wall defect for eight dogs (approximately 5×12 cm). Themesh edges must surpass the edges of the abdominal defect with at least1 cm. An omental pedicle was retrieved and fixed with individual stit-ches of polypropylene 2/0 suture material (Prolene-Ethicon, Germany)to the perimeter of the mesh, leaving adequate strands which were thenanchored to the perimeter of the abdominal wall defect. Mosquitoforceps were used to clamp the suture strands to keep them in positionbefore being tied when the mesh was in place to avoid any tension orwrinkles. Few stitches of the same suture material were placed alongthe perimeter of the defect with the mesh for better fixation and sta-bilization (Fig. 2c). The subcutaneous abdominal fascia was closed withcontinuous suture pattern using Poygalactin 2/0 (Vicryl-Ethicon, Ger-many) before closing the skin with simple interrupted suture usingProlene 2/0.

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2.5.4. Retroperitoneal mesh hernioplastyThe developed meshes were fixed to the perimeter of the induced

abdominal wall defects for four dogs using Prolene 2/0 (Fig. 2d). Thesubcutaneous abdominal fascia and the skin were closed as previouslymentioned.

2.5.5. Post-operativePost-operative care comprised isolation in separate boxes and ad-

ministration of antibiotics for 7 days and analgesic (Carprofen 5%,50mg, Adwia, Egypt) for 3 days. Restriction of diet to half the amountfor 3 days was done. The operated animals were kept under clinicalobservation to record any local reaction or complications in terms ofdehiscence of the skin wound, mesh incompatibility and infection orsinus formation.

2.5.6. Histopathological evaluationRetroperitoneal mesh hernioplasty dogs and intraperitoneal mesh

hernioplasty dogs were euthanized at one month and two months afteroperation to evaluate gross wound adhesion or any signs of infection ormesh prolapse. Tissue samples from soft healed wounds with remnantsof mesh were processed for histopathology and stained by hematoxylenand eosin (Suvarna, Layton, & Bancroft, 2012). Adhesion between themesh and intraabdominal organs were evaluated by scoring system,where zero-score represents thin or narrow and easily separable adhe-sions, one-score represents adhesions limited to a small area, two-scorerepresents thick adhesions dispersed over a large area, three-score re-presents thick and wide adhesions as well as adhesions of the organs tothe anterior and/or posterior abdominal wall, and finally, four-scorerepresents migration of the prosthetic material and presence of entero-cutaneous fistula (Mazuji, 1964). On the other hand, fibroblast densityscoring system was used as follows: zero-score represents absence offibrosis, one-score represents minimal loose fibrosis, two-score re-presents moderate fibrosis, and three-score represents florid and mas-sive fibrosis (Hooker, Taylor, & Driman, 1999). Finally, the used

inflammation scoring system was: zero-score represents no inflamma-tion, one-score represents large cells as well as rare/dispersed lym-phocytes and plasma cells, two-score represents large cells togetherwith increased number of lymphocytes, neutrophils and plasma cells,three-score represents multiple mixed inflammatory cells and presenceof micro-abscess (Hooker et al., 1999).

2.6. Statistical analysis

Data were expressed in terms of mean ± standard deviation.Significant difference tests were used like one-way analysis of variance(ANOVA) (*p < 0.05, **p < 0.01, ***p < 0.001 and****p < 0.0001) using the software GraphPad Prism Software Version6.

3. Results and discussion

Over the last decades, the surgical procedures for hernia repair havebeen standardized. However, surgeons and researchers worldwide arefocusing on conventional mesh-based technology. Today, severalprosthetic materials of different natures were tested. In the currentstudy, a modification to the standard polyester fabric is proposed,where well-selected natural polymers and drugs were used to improvethe prosthetic mesh implantation outcome.

3.1. Preparation and characterization of PH-loaded PL nanomicelles

Pluronic F-127 (PL) is a triblock amphiphilic copolymer with hy-drophobic core of poly(propylene oxide) and hydrophilic tails of poly(ethylene oxide). The special structure of PL allows the formation ofself-assembled nanomicelles, and during assembly of the micelles, hy-drophobic drugs like PH could be encapsulated efficiently (Fig. 3a). Themicelles characteristics are dependent on the drug to polymer ratio.Therefore, in the current study three ratios of drug: polymer (1:2, 1:4

Fig. 2. In-vivo abdominal wall defect dog model: (a) a schematic representation of surgery procedures, (b) abdominal wall defect, (c) intraperitoneally im-plantation, and (d) retroperitoneal implantation.

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and 1:8) were investigated. Particle size was measured using DLStechnique, where a significant decrease from 130 ± 13 nm to72.3 ± 3 nm was observed upon increasing the polymer (PL) ratio(Fig. 3b). This could be attributed to the assembly of more micelles,where the drug (PH) works as a nucleus that attract the hydrophobicparts of polymer. Several studies were focused on studying the effect ofdrug hydrophobicity on the nanomicelle formation. For instance, theeffect of hydrophobicity of ibuprofen, aspirin and erythromycin werestudied by Basak & Bandyopadhyay (2013). Moreover, this observationwas previously reported in our previous study, where particle sizesignificantly decreased when PH was loaded in the PL nanomicelles (Aliet al., 2016). Also, our group reported the encapsulation of azi-thromycin in PL nanomicelles with particle size around 90 nm, whichproves the effect of drug nature (Khalil et al., 2019). Furthermore,particle size homogeneity was observed, and the polydispersity index(PDI) significantly decreased when the polymer ratio increased, where1:8 ratio demonstrated only 0.33 PDI (Fig. 3c). It is worth to mentionthat the hydroxyl end groups of PL are dominating its overall charge,which previously reported as -15.7 ± 1 (Ali et al., 2016). In the currentstudy, loading of PH has increased the surface negativity to−17.1 ± 1without statistically significant difference (Fig. 3d). Furthermore, in-creasing the polymer ratio slightly increased the PL surface negativityto −20.1 ± 3. This observation was previously reported for azi-thromycin, where the negativity increased from −14.5 to −15.4 innanomicelles of drug: polymer ratio of 1:1 and 1:3, respectively (Khalilet al., 2019). The morphology of resulting PL nanomicelles was de-monstrated in Fig. 3e. As apparent from the figure, spherical shapeparticles were observed with a slight heterogeneity. Thermal analysiswas carried out to confirm drug (PH) encapsulation in the developed PLnanomicelles as shown in Fig. 3f. DSC thermograms demonstrated thedistinctive endothermic peaks at 297.14⁰C and 60.6⁰C that correspondto the melting points of free PH and PL, respectively (Ali et al., 2016). Inthe thermogram of the investigated PH-loaded PL nanomicelles (P3),PH peak has disappeared which confirms that the drug was successfullyloaded within the PL matrix. As also apparent from the figure (Fig. 3g),the entrapment efficiency (EE%) increased significantly in the case of1:4 and 1:8 ratio as compared with 1:1 ratio. Furthermore, no sig-nificant difference was found between 1:4 and 1:8 ratios. This makesthe nanomicelles formulation with the 1:4 ratio a good candidate,where the EE% reached around 90% and the drug loading (LD%) wasabout 20% (Fig. 3h). The release profile of PH was measured in

hydroalcoholic PBS (Fig. 3i). Plain drug (PH) was completely dissolvedwithin 6 h. On the other hand, the encapsulated PH in all ratios wasreleased within 48 h. The PL nanomicelles formulation of 1:8 ratio (P3)demonstrated a slight increase in the PH release than noted in the othertwo formulations (P1 and P2). The release profile was best fitted toKorsmeyer-Peppas kinetic model with r2 > 0.94 for all ratios. Thediffusional exponent value (n) was equal to 0.33 which represents afickian diffusion model (Costa & Sousa Lobo, 2001).

3.2. Preparation and characterization of CPX-ALG microcomplexes

Oppositely charged molecules when presented together in aqueoussolutions can interact spontaneously to form polyelectrolyte complexes(Sæther, Holme, Maurstad, Smidsrød, & Stokke, 2008). The polyelec-trolyte complex could be formed from two counterparts of differentnatures. For instance, CS and ALG as oppositely charged biopolymerswere complexed together as reported by Sæther et al. (2008). Also, thenegatively charged sodium tripolyphosphate (TPP) was reported asionic crosslinker for the cationic CS chains, and their interaction pro-duct could be described as a polyelectrolyte complex (Ramasamy et al.,2014). Moreover, polyelectrolyte complexes could be formed between acharged drug like CPX and an anionic surfactant such as sodium do-decyl sulfate, which was used to improve drug loading efficiencies inpoly(lactic-co-glycolic acid) (PLGA) NPs (Günday Türeli, Türeli, &Schneider, 2016). Our group was developed and investigated, in aprevious study, polyelectrolyte complexes based on doxorubicin andALG followed by coating with a CS nanolayer, and this approach hasimproved the drug loading percentage (Hefnawy, Khalil, & El-Sherbiny,2017). In the current study, CPX formed a polyelectrolyte complexesmicroparticles with ALG (Fig. 4a). CPX contains two functional groups,the carboxylic acid and the piperazine group. At the pH values lowerthan 6 (pKa1), the soluble cationic form (CPX+) is the predominant dueto protonation of the amine group in the piperazine moiety. On theother hand, ALG consists of an anionic chain of (1–4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) in different arrange-ments and proportions. The CPX-ALG complexes were formed with aparticle size of 2549 ± 280 nm (Fig. 4b). Gradual increase of the ALGratio significantly decreased the particle size which reached1292 ± 99 nm at 1:8 drug to polymer ratio (Fig. 2b). This could beattributed to the crosslinking effect of CPX to the ALG polymer chains.Changing the CPX: ALG ratio led to formation coiled chain structure

Fig. 3. Phenytoin (PH)-loaded PL nanomi-celles: (a) diagram illustrating the nanomicellestructure, (b) particle size as measured by DLS,(c) polydispersity index (PDI), (d) zeta poten-tial, (e) scanning electron microscope micro-graph, (f) thermal analysis using DSC, (g) en-trapment efficiency (EE%), (h) drug loading(LD%), and (i) the in-vitro release profile.

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followed by pregel state to form a small ALG nucleus (Sarmento,Ribeiro, Veiga, Ferreira, & Neufeld, 2007). Moreover, ALG is a well-known polymer with fast crosslinking behavior, which makes it difficultto control particle homogeneity. This was observed in Fig. 2c, wherethe PDI was around 0.8. Furthermore, increasing ALG ratio increasedsignificantly the surface negativity of the developed CPX-ALG micro-particles to −20.19 (Fig. 4d). The thermal analysis of CPX using DSC(Fig. 4e) demonstrated two characteristic endothermic peaks at205.1 °C and 345.3 °C beside one exothermic peak noted at 354.3 °C(Tan, Tan, Zhao, & Li, 2012). On the other hand, a single endothermicpeak was observed at 101.8 °C in the case of ALG thermogram. Thisendothermic peak is usually referred to the loss of water from the hy-drophilic polymer. Also, an exothermic peak was observed in the ALGthermogram which can be correlated to the partial decarboxylation ofthe polymer (Tsang et al., 2015). The thermogram of formed CPX-ALGcomplex (C4) demonstrated all the characteristic peaks of both CPX andALG, which proves the success of polyelectrolyte complex formation.

The complexation efficiency was measured for all the formedcomplexes as shown in Fig. 4f. All complexes showed high complexa-tion efficiency which exceeded 80%. Increasing the ALG ratio sig-nificantly decreased the drug (CPX) loading percentage (Fig. 4g). Onthe other hand, the release profile of CPX:ALG 1:1 (C1) and CPX:ALG1:8 (C4) were measured (Fig. 4h). Both complexes showed similar re-lease profiles, which extended to three days. The release profile wasbest fitted to Korsmeyer-Peppas kinetic model with r2 > 0.98 for allratios. The diffusional exponent value (n) was equal to 0.277 whichrepresents a fickian diffusion model (Costa & Sousa Lobo, 2001).

Staphylococcus aureus (gram-positive bacteria) and Pseudomonasaeruginosa (gram-negative bacteria) were used to determine the MICusing microdilution test (Fig. 4i). MIC of CPX was 0.5 μg/ml againstStaphylococcus aureus (Maleki Dizaj, Lotfipour, Barzegar-Jalali,Zarrintan, & Adibkia, 2017), and the complexation of CPX with ALG hassignificantly decreased this MIC to 0.25 μg/ml. To the same extent, MIC

of CPX was 0.25 μg/ml against Pseudomonas aeruginosa (Garhwal et al.,2012), and the complexation of CPX has significantly improved theantimicrobial activity with a MIC value of 0.125 μg/ml.

3.3. Preparation of the drugs-loaded CS coating

CS-based films were extensively used in different applications owingto the nature of CS being biodegradable and biocompatible poly-saccharide. Besides, its cationic nature allows the formation of gels andfilms, where electrostatic interactions with polyanions, such as TPP orALG, can physically crosslink the CS chains. TPP is one of the commonpolyanions that used as a crosslinker for CS films (Shu & Zhu, 2002). Inspite of the desirable biological properties of CS, its poor mechanicalcharacteristics has limited its use in abdominal hernia, where enoughresistance to mechanical strains is one of the main features that must beavailable in prosthetic meshes. Therefore, commercial polyester fabric(CPF) of known good mechanical properties (Fig. 5a) was used as thesubstrate, and has been incorporated into CS film in form of a sandwich-like structure. CS-coated CPF was successfully prepared as shown inFig. 5b. Also, loading of CPX-ALG microparticles and the PH-PL nano-micelles into the CS coating layer was successfully achieved as shown inFig. 5c and d. Thermal analysis revealed the formation of CS film,where the CS endothermic peak at 100.3 °C was observed (Fig. 5e). Therelease profile of PH from CPF2 showed a slight decrease in drug re-lease, which could be attributed to the effect of CS-crosslinked matrix inCPF2 film. The kinetic fitting model was found to be Korsmeyer-Peppaswith r2 > 0.95. The diffusional exponent value (n) was equal to 0.31which represents a fickian diffusion model (Fig. 5f). On the other hand,the release profile of CPX from CPF3 also showed a slight decrease indrug release. The kinetic fitting model was also found to be Korsmeyer-Peppas kinetic model with r2 > 0.98. The diffusional exponent value(n) was equal to 0.4 which represents a fickian diffusion model (Fig. 5f).

Fig. 4. Ciprofloxacin (CPX)-alginate (ALG) microcomplexes: (a) a diagram illustrating the formed microcomplexes, (b) particle size as measured by DLS, (c)polydispersity index (PDI), (d) zeta potential, (e) thermal analysis as measured by DSC, (f) complexation efficiency, (g) drug loading, (h) in-vitro release profile, and(i) the in-vitro microbiological study.

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3.4. In-vivo study

Hernioplasty is one of the most common procedures in surgicalfield. Generally, the management of hernia defect using mesh is thestandard technique. Currently, researchers are working on developingnew materials that could improve the surgical outcomes. In the currentstudy, two mesh hernioplasty techniques were conducted; in-traperitoneal implantation and retroperitoneal implantation. All ani-mals had an ordinary recovery without clinical signs of wound dehis-cence, herniation or infection. Only 3 animals showed mild edema withexcessive accumulation of serous fluid during the first week after sur-gery, which gradually reduced as healing progressed. One animal afterone week exhibited a small subcutaneous abscess which was openedand daily dressed with povidone iodine till complete healing after 10days.

After one and two months, gross morphological examination of theabdominal wall at the implantation site revealed unaltered integrity ofthe CPF meshes, which were uniformly infiltrated with connectivetissue of variable thickness (Fig. 6a–d). Furthermore, no detectableadhesions or rejection signs were observed except one case in group Bretroperitoneal implantation, which exhibited partial mesh prolapse.On the other hand, all cases of intraperitoneal prosthetic mesh im-plantation with omentalization were healed without any visceral ad-hesions (zero-score), which were limited between the omental pedicleand the visceral surface of the CPF mesh. This was in agreement withprevious reposts (Aydinli et al., 2007; Shoukry et al., 1997). In thisrespect, omentopexy acting as a barrier so that adhesion with the vis-cera could be avoided. Furthermore, all dogs treated with CS-coatedCPF prosthetic mesh (groups B–D) showed almost a complete de-gradation and uniformly infiltrated by fibrous connective tissue, whichfirmly incorporated into the abdominal wall upon histological ex-amination at 2-month post-operation. These observations prove thesuccess of the modified CPF as an ideal and feasible alternative pros-thesis with many advantages like low cost, inertness, biocompatibility,mechanical stability, pliability, low infection rate, limited modificationby body tissues, sterilizability, non-carcinogenicity, limited

inflammatory reaction, hypoallergenic and minimal complications(Salgaonkar & Lomanto, 2017; Silverman, Li, Holton, Sawan, &Goldberg, 2004).

Microscopic examination of tissue sections of abdominal wallmuscles and subcutaneous fascia of group A (control) showed remnantsof mesh fibers surrounded by intense leukocytic infiltration mainlyneutrophils, lymphocytes and macrophages with focal scattered micro-abscess (Fig. 6e and f). Minimal loose fibrous connective tissue pro-liferation with focal areas of myxomatous degeneration was noted(Fig. 6g). In group B (non-medicated CS-coated mesh), tissue sectionsshowed a marked reduction of remnants of mesh fibers and similarleukocytic infiltration but with few neutrophils. Numerous numbers ofmultinucleated foreign bodies giant cells were also seen (Fig. 6h and i).Moderate fibrous connective tissue proliferation was noticed (Fig. 6j).In group C (antibacterial drug, CPX, incorporated CS-coated mesh),tissue sections showed few remnants of mesh fibers and leukocytic in-filtration with few numbers of small multinucleated giant cells (Fig. 6kand l). Moderate fibrous connective tissue proliferation was also no-ticed (Fig. 6m). In group D (both drugs-loaded CS-coated mesh), tissuesections showed scares remnants of mesh fibers surrounded by densefibrous tissue and mononuclear cells mainly lymphocytes and macro-phages with few numbers of giant cells (Fig. 6n and o). On the otherhand, massive fibrous connective tissue proliferation was noticed(Fig. 6p). The recorded histological scoring of fibroblast density andinflammation are illustrated in Fig. 6q–u.

4. Conclusion

This study proposed and evaluated the effect of coating commercialpolyester fabric with natural biomaterial incorporating nanocarriersloaded with antibacterial drug (ciprofloxacin) and healing promotor(phenytoin). Overall, an efficient healing with excellent biocompat-ibility were achieved, where the new mesh platform is an ideal andfeasible alternative prosthesis with many advantages like low cost, in-ertness, biocompatibility, mechanical stability, pliability, low infectionrate, limited modification by body tissues, sterilizability, non-

Fig. 5. Chitosan (CS)-coated commercial polyester fabric film: (a) commercial polyester fabric image, (b) CS-coated commercial polyester fabric image, (c) CS-coated commercial polyester fabric loaded with CPX-ALG complex microparticles, (d) CS-coated commercial polyester fabric loaded with PH-PL nanomicelles andCPX-ALG microparticles, (e) thermal analysis, and (f) in-vitro release profile.

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carcinogenicity, limited inflammatory reaction, hypoallergenic andminimal complications. It is also worthy to mention here that, in-traperitoneal pedicled omentopexy is recommended to prevent visceraladhesion to the used mesh. Our future studies will investigate thecoating of different types of synthetic meshes with the coating platformdeveloped in the present study as well as studying the efficiency of thefabricated hybrid prosthetic mesh on various types and grades ofhernia.

Funding

No funding was received.

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